Human astroviruses (H-Ast) are the prototype members of an important new family of RNA viruses that infect the gastrointestinal tract. The 6.8 kb positive sense RNA genome of H-Ast is organized with nonstructural proteins encoded at the 5' end by ORFs 1a and 1b, and the capsid protein(s) by ORF-2 at the 3' end. Sequence analysis indicates that ORF-1a encodes a 3C-like serine protease motif and a nuclear localization signal (NLS). ORF-1b encodes an RNA-dependent RNA polymerase (RdRp) motif, and its translation is dependent on (-1) ribosomal frameshifting at a specific signal found in the overlap region between ORF-1a and -1b. By this mechanism, the RdRp is expressed as part of a 1a/1b polyprotein that must be processed further to be functional. Our project focuses on determining how the viral NSPs are processed into functional subunits and where they are localized within the infected cell. We hypothesize that the putative viral 3C-like serine protease, encoded by ORF- I a, is responsible for cleavage of the ORF-1 a encoded product and 1a/1b polyprotein into functioning subunits, and that some of these cleavage products are directed to the nucleus by a functional NLS that is part of the 1a protein. Our specific aims are: 1) characterizating the role of the virally encoded protease in processing of the nonstructural gene products and 2) characterizing the subcellular localization of the viral nonstructural proteins. These studies will provide significant new information on the processing of the H-Ast nonstructural proteins and their subcellular localization. These data should contribute to our understanding of viral replication and ultimately, viral pathogenesis.